Since the demonstration of Borrelia burgdorferi as the infectious agent of Lyme borreliosis (Burdorfer, W. et at. 1982. Science 216:1317-1319), numerous studies have documented the difficulty of culturing the spirochetes from or observing spirochetes in infected mammalian hosts (Steere, A. C. 1989. New Engl. J. Med. 321: 586-596; Szczepanski, A. et al. 1991. Microbiol. Rev. 55: 21-34). Factors such as the reportedly sparse distribution of B. burgdorferi in hosts, the fastidious growth requirements, and the relatively slow growth rate of this spirochete compound the problems associated with aseptic primary isolations.
The immunological interactions between the Lyme disease spirochete, Borrelia burgdorferi, and its mammalian hosts are poorly understood (Bosler, E. M., D. P. Cohen, T. L. Schulze, C. Olsen, W. Bernard, and B. Lissman. 1988. Host responses to Borrelia burgdorferi in dogs and horses, p. 221-234. In J. L. Benach, and E. M. Bosler (eds.), Lyme disease and related disorders. Annals of the New York Academy of Sciences 539; Dlesk, A., D. F. Bjarnason, P. Mitchell, and P. McCarty. 1988. Lyme disease presenting as seronegative rheumatoid arthritis, p. 454-455. In J. L. Benach, and E. M. Bosler (eds.), Lyme disease and related disorders. Annals of the New York Academy of Sciences 539; Duray, P. H. and A. C. Steere. 1988. Clinical pathologic correlations of Lyme disease by stage, p. 65-79. In J. L. Benach, and E. M. Bosler (eds.), Lyme disease and related disorders. Annals of the New York Academy of Sciences 539; Fox, J. L. 1989. Interest in Lyme disease grows. ASM News 55:65-66; Hyde, F. W., R. C. Johnson, T. J. White, and C. E. Shelburn. 1989. Detection of antigen in urine of mice and humans infected with Borrelia burgdorferi, etiologic agent of Lyme disease. J. Clin. Microbiol. 27:58-61; Magnarelli, L. A. 1988. Serologic diagnosis of Lyme disease, p. 154-161. In J. L. Benach, and E. M. Bosler (eds.), Lyme disease and related disorders. Annals of the New York Academy of Sciences 539; Schwan, T. G., W. Burgdorfer, and C. F. Garon. 1988. Changes in infectivity and plasmid profile of the Lyme disease spirochete, Borrelia burgdorferi, as a result of in vitro cultivation. Infect. Immun. 56:1831-1836; Schwan, T. G., W. Burgdorfer, M. E. Schrumpf, and R. H. Karstens. 1988. The urinary bladder, a consistent source of Borrelia burgdorferi in experimentally infected white-footed mice (Peromyscus leucopus). J. Clin. Microbiol. 26:893-895; Sticht-Groh, V., R. Martin, and I. Schmidt-Wolf. 1988. Antibody titer determinations against Borrelia burgdorferi in blood donors and in two different groups of patients, p. 497-499. In J. L. Benach, and E. M. Bosler (eds.), Lyme disease and related disorders. Annals of the New York Academy of Sciences 539; Stiernstedt, G., R. Gustafusson, M. Kaarlsson, B. Svenungsson, and B. Skoldenberg. 1988. Clinical manifestations and diagnosis of neuroborreliosis, p. 46-55. In J. L. Benach, and E. M. Bosler (eds.), Lyme disease and related disorders. Annals of the New York Academy of Sciences 539 and Wilske, B., V. Preac-Mursic, G. Schierz, R. Kuhbeck, A. G. Barbour, and M. Kramer. 1988. Antigenic variability of Borrelia burgdorferi. p. 126-143. In. J. L. Benach, and E. M. Bosler (eds.), Lyme disease and related disorders. Annals of the New York Academy of Sciences 539). Most mammalian hosts mount an antibody response to the spirochete, however the antibodies are often serologically cross-reactive with other species of Borrelia, and individuals with sero-negative infections have been encountered using standard screening criteria (Dlesk, A., D. F. Bjarnason, P. Mitchell, and P. McCarty. 1988. Lyme disease presenting as seronegative rheumatoid arthritis, p. 454-455. In J. L. Benach, and E. M. Bosler (eds.), Lyme disease and related disorders. Annals of the New York Academy of Sciences 539; Fox, J. L. 1989. Interest in Lyme disease grows. ASM News 55:65- 66; Hyde, F. W., R. C. Johnson, T. J. White, and C. E. Shelburn. 1989. Detection of antigen in urine of mice and humans infected with Borrelia burgdorferi, etiologic agent of Lyme disease. J. Clin. Microbiol. 27:58-61; Magnarelli, L. A. 1988. Serologic diagnosis of Lyme disease, p. 154-161. In J. L. Benach, and E. M. Bosler (eds.), Lyme disease and related disorders. Annals of the New York Academy of Sciences 539; Sticht-Groh, V. R. Martin, and I. Schmidt-Wolf. 1988. Antibody titer determinations against.Borrelia burgdorferi in blood donors and in two different groups of patients, p. 497-499. In J. L. Benach, and E. M. Bosler (eds.), Lyme disease and related disorders. Annals of the New York Academy of Sciences 539 and Wilske, B., V. Preac-Mursic, G. Schierz, R. Kuhbeck, A. G. Barbour, and M. Kramer. 1988. Antigenic variability of Borrelia burgdorferi. p. 126-143. In. J. L. Benach, and E. M. Bosler (eds.), Lyme disease and related disorders. Annals of the New York Academy of Sciences 539:126-143). Furthermore, strain variation among B. burgdorferi isolates, and antigenic variation within populations render immunodiagnostics, based on monoclonal antibodies, potentially insensitive and unreliable for detection of circulating and excreted antigens in some hosts (Barbour, A. G., R. H. Heiland, and T. R. Howe. 1985. Heterogeneity of major proteins in Lyme disease borreliae: a molecular analysis of North American and European isolates. J. Infect. Dis. 152:478-484 and Wilske, B., V. Preac-Mursic, G. Schierz, R. Kuhbeck, A. G. Barbour, and M. Kramer. 1988. Antigenic variability of Borrelia burgdorferi. p. 126-143. In. J. L. Benach, and E. M. Bosler (eds.), Lyme disease and related disorders. Annals of the New York Academy of Sciences 539:126-143). Therefore, clinical symptoms, patient history and occasional primary isolations of the spirochete from blood or tissue biopsies, provide the bases for most diagnoses (Benach, J. L., E. M. Bosler, J. P. Hanrahan, J. L. Coleman, G. S. Habicht, T. F. Bast, D. J. Cameron, J. L. Ziegler, A. G. Barbour, W. Burgdorfer, R. Edelman, and R. A. Kaslow. 1983. Spirochetes isolated from the blood of two patients with Lyme disease. N. Engl. J. Med. 308:740-742; Dlesk, A., D. F. Bjarnason, P. Mitchell, and P. McCarty. 1988. Lyme disease presenting as seronegative rheumatoid arthritis, p. 454-455. In J. L. Benach, and E. M. Bosler (eds.), Lyme disease and related disorders. Annals of the New York Academy of Sciences 539:454-455; Duray, P. H., and A. C. Steere. 1988. Clinical pathologic correlations of Lyme disease by stage, p. 65-79. In J. L. Benach, and E. M. Bosler (eds., Lyme disease and related disorders. Annals of the New York Academy of Sciences 539:65-79 and Rawlings, J. A., P. V. Fornier, and G. J. Teltow. 1987. Isolation of Borrelia spirochetes from patients in Texas. J. Clin. Microbiol. 52:1148-1150.20). Such problems are often cited as factors influencing the reportedly poor diagnostic acumen for Lyme disease (Fox, J. L. 1989. Interest in Lyme disease grows. ASM News 55:65-66).
Considerable work is currently directed toward identifying conserved, species-specific cell surface antigens for diagnostic use, and for epidemiological and pathogenetic studies. Expression of outer surface protein A (OspA) is considered universal among B. burgdorferi isolates, but not among related spirochetes (Barbour, A. G., R. H. Heiland, and T. R. Rowe. 1985. Heterogeneity of major proteins in Lyme disease borreliae: a molecular analysis of North American and European isolates. J. Infect. Dis. 152:478-484; Barbour, A. G., S. L. Tessier, and W. J. Todd. 1983. Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody. Infect. Immun. 41:795-804; Bergstrom, S., V. G. Bundoc, and A. G. Barbour. 1989. Molecular analysis of linear plasmid-encoded major surface proteins, OspA and OspB, of the Lyme disease spirochaete Borrelia burgdorferi. Mol. Microbiol. 3:479-486; Hyde, F. W., R. C. Johnson, T. J. White, and C. E. Shelburn. 1989. Detection of antigen in urine of mice and humans infected with Borrelia burgdorferi, etiologic agent of Lyme disease. J. Clin. Microbiol. 27:58-61; Magnarelli, L. A. 1988. Serologic diagnosis of Lyme disease, p. 154-161. In J. L. Benach, and E. M. Bosler (eds.), Lyme disease and related disorders. Annals of the New York Academy of Sciences 539:154-161 and Wilsek, B., V. Preac-Mursic, G. Schierz, R. Kuhbeck, A. G. Barbour, and M. Kramer. 1988. Antigenic variability of Borrelia burgdorferi, etiologic agent of Lyme disease. J. Clin. Microbiol. 27:58-61 and Wilske, B., V. Preac-Mursic, G. Schierz, R. Kuhbeck, A. G. Barbour, and M. Kramer. 1988. Antigenic variability of Borrelia burgdorferi p. 126-143. In J. L. Benach, and E. M. Bosler (eds.), Lyme disease and related disorders. Annals of the New York Academy of Sciences 539:126-143). This protein is immunogenic, however, surface-exposed regions appear to be antigenically variable, since surface-reactive monoclonal antibodies to OspA fail to recognize some isolates (Barbour, A. G., R. H. Heiland, and T. R. Howe. 1985. Heterogeneity of major proteins in Lyme disease borreliae: a molecular analysis of North American and European isolates. J. Infect. Dis. 152:478-484; Hyde, F. W., R. C. Johnson, T. J. White, and C. E. Shelburn. 1989. Detection of antigen in urine of mice and humans infected with Borrelia burgdorferi. p. 126-143. In. J. L. Benach, and E. M. Bosler (eds.), Lyme disease and related disorders. Annals of the New York Academy of Sciences 539:126-143).
Recent experiments have shown that OspA and several other proteins are exported from B. burgdorferi cells in membrane vesicles (Garon, C. F., D. W. Dorward, and M. D. Corwin. 1989. Structural features of Borrelia burgdorferi-- Lyme disease spirochete: silver staining for nucleic acids. Scanning Microscopy Supplement 3, pages 109-115; Dorward, D. W., T. G. Schwan, and C. F. Garon, 1991. J. Clin. Microbiol. 29:1162-1171). Indirect evidence suggests these vesicles may be produced by spirochetes in vivo providing sustained antigenic challenge to hosts maintaining a limited population of spirochetes (Fox, J. L. 1989. Interest in Lyme disease grows. ASM News 55:65-66). To determine whether B. burgdorferi vesicles occur in experimentally-infected mice, polyclonal rabbit sera were generated against vesicles and an 83 kilodalton (kDa), major extracellular protein (MEP). Using these reagents, a 2-stage immune electron-microscopic assay was developed for first capturing then identifying extracellular B. burgdorferi infections.
The closest known technology to the present invention was a diagnostic kit, initially produced by 3M Corp. (Fast Lyme, Cat. No. 700-500) that used monoclonal antibodies to detect Lyme antigen in human urine samples. The kit was limited to urine samples, provided false-negative results with geographically diverse samples, and was marginally sensitive (Hyde F. W. et al, (1989), Detection of antigen in urine of mice and humans infected with Borrelia burgdorferi, etiologic agent of Lyme disease, J. Clin. Microbiol. 27:58-61). Moreover, the kit technology, owned by BioWhittaker, is neither FDA approved for human testing nor currently marketed.